Biochemistry 1995, 34, 11963- 11969
11963
W-Cyanoborane-2'-Deoxyguanosine 5'-Triphosphate Is a Good Substrate for DNA Polymerase? Kenneth W. Porter, Jeno Tomasz, Faqing Huang, Anup Sood, and Barbara Ramsay Shaw* Department of Chemistry, Duke University, Durham, North Carolina 27708 Received March 7, 1994@
The 5'-triphosphate of the boronated nucleoside analog N7-cyanoborane-2'-deoxyguanosine (7bdGTP)was synthesized, and a series of experiments was initiated to assess the potential of the compound to serve as a substrate for DNA polymerases. We show here that 7bdGTPcan be incorporated into DNA by Sequenase. The resulting hemiboronated extension products are resistant to cleavage by treatment with either DMS and heat or a number of restriction enzymes. Further, in the polymerase chain reaction, 7bdGTPcan be utilized as a substrate for Tuq polymerase. Finally, by kinetic analysis, we have found that 7bdGTP is a more efficient substrate for exonuclease-free Klenow than normal dGTP. Thus, the introduction of a cyanoborane moiety to the N7 position of dGTP results in a nucleotide that is accepted in lieu of normal dGTP by a number of DNA polymerases. ABSTRACT:
Our laboratory has synthesized a new class of modified 2'-deoxynucleosides that introduce a cyanoborane moiety to the purine bases of 2'-deoxyguanosine 1, 2'-deoxyinosine 2, and 2'-deoxyadenosine 3 and to the pyrimidine base of 2'-deoxycytidine 4 (Sood et al., 1989). Preliminary in vitro and in vivo studies in rodents have shown that cyanoboronated nucleosides possess a number of potentially useful pharmacological properties, including antineoplastic (Sood et al., 1992), hypolipidemic (Hall et al., 1993), and antiinflammatory activities (Rajendran et al., 1994). 0
CN
OH
OH
ndG
7bd~
1
2
OH
OH
lbdA
3bdC
3
4
The pharmacological activity of a number of 2'-deoxynucleoside analogs depends on the ability of the compound Boron-Containing Nucleic Acids, Part 4. This work was supported by Grant NP-741 from the American Cancer Society to B.R.S. and a subgrant of Department of Energy Grant DE-FG05-90ER81065 to B.R.S. from Boron Biologicals, Inc. * Author to whom correspondence should be addressed. Abstract published in Advance ACS Abstracts, July 15, 1994. @
0006-2960/95/0434-11963$09.00/0
to be phosphorylated by cellular kinases and subsequently incorporated into DNA by DNA polymerases (Reid et al., 1988; Reardon & Spector, 1989; Reardon, 1992). Studies by Sood et al. (1992) suggest that phosphorylation and subsequent incorporation into DNA is a possible mechanism for the antineoplastic activity of the cyanoboronated nucleosides. Those results showed that M-cyanoborane-2'-deoxycytidine (4, 3bdC1)was taken up rapidly by Tmolt3 leukemia cells. After 24 h, a significant amount of the 3bdCwas found to be associated with DNA and/or RNA fractions of the Tmolt3 leukemia cells. Of the major cyanoboronated deoxynucleosides,the deoxyguanosine derivative (1) is the only compound in which the cyanoborane moiety does not interfere with the potential Watson-Crick hydrogen-bonding groups. Preliminary physical studies by the method of Williams et al. (1989) have shown that ~-cyanoborane-2'-deoxyguanosine (7bdG,(1)) is capable of base-pairing with 2'-deoxycytidine (B. Banks and B. R. Shaw, unpublished results). Therefore, it is possible that after cellular uptake and phosphorylation, the cyanoboronated deoxyguanosine triphosphate may be recognized by DNA polymerase and inserted into DNA opposite dC. In this paper, we report the synthesis of the 5'-triphosphate of the boronated 2'-deoxyguanosine nucleoside (M-cyanoborane-2'-deoxyguanosine 5'-triphosphate, Le., 7bdGTP (S)), I Abbreviations: 3bdC,Nkyanoborane-2'-deoxycytidine; 7bdG,A"cyanoborane-2'-deoxyguanosine; dG, 2'-deoxyguanosine; 7bdGTP,A"cyanoborane-2'-deoxyguanosine 5'-triphosphate; dGTP, 2'-deoxyguanosine 5'-triphosphate; dA, 2'-deoxyadenosine; dT, thymidine; dC, 2'deoxycytidine; dI, 2'-deoxyinosine; bp, base pairs; CD, circular dichroism; CM, carboxymethyl; DNA, 2'-deoxyribonucleic acid; DEAE, (diethy1amino)ethyl; DMS, dimethyl sulfate; EDTA, ethylenediaminetetraacetic acid; HPLC, high-performance liquid chromatography; Klenow, the large fragment of Escherichia coli DNA polymerase I; MS, mass spectrometry; NMR, nuclear magnetic resonance; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT, room temperature; Tris, tris(hydroxymethy1)aminomethane; UV, ultraviolet.
0 1995 American Chemical Society
11964 Biochemistry, Vol. 34, No. 37, 1995 and an analysis of its incorporation into DNA by polymerases. Incorporation was performed using three prokaryotic DNA polymerases: Sequenase, Taq DNA polymerase, and an exonuclease-free mutant of the Klenow fragment from Escherichia coli DNA polymerase I. Sequenase and Taq polymerase were able to insert 7bdGTPinto DNA and extend the growing strand past the point of insertion. Exonucleasefree Klenow was used to determine the kinetic parameters for 7bdGTPincorporation. Results show that 7bdGTPwas incorporated into DNA more efficiently than normal dGTP.
OH
5
MATERIALS AND METHODS Materials. N,N-Dimethylformamide was distilled from CaH2. Trimethyl phosphate (Aldrich, 99%) was distilled at 1 mmHg and stored over 4-8, molecular sieves. Phosphoryl chloride was distilled and stored in sealed ampules at 4 "C. Bis(tri-n-butylammonium) pyrophosphate (0.5 M) in N,Ndimethylformamide was prepared as described by Tomasz (1983). DEAE-cellulose (DE-32) and CM-cellulose (CM32) were purchased from Whatman. Kieselgel 60 F254 0.2mm chromatoplates were purchased from Merck. Oligonucleotides were synthesized on an AB1 380B DNA synthesizer, purified by denaturing PAGE, and recovered by electroelution. Sequenase version 2.0 (modified T7 DNA polymerase) and exonuclease-free Klenow were purchased from USB. Taq polymerase was purchased from Cetus. M13mp2 DNA was provided kindly by Ted Gonzalez (Frederico et al., 1990). [ Y - ~ ~ P I Aand T P [ y 3 9 ] A T P (> 1000 Ci/mmol) were purchased from Amersham. NMR. NMR spectra of an approximately 0.1 M solution were recorded at ambient temperature on a Varian Associates Model XL-300 spectrometer at 300 (IH), 121.44 (31P), or 75.57 MHz (13C). 'H and 13C chemical shifts were referenced to tetramethylsilane, while 31Pshifts were referenced to 85% H'jP04. Mass Spectrometry. MS spectra were taken on a VG 70s tandem hybrid MSMS spectrometer run in the MS only mode. The accelerating voltage was 10 kV. A cesium ion gun at 35 kV was used for sample ionization using glycerol as a matrix. Evaporations were carried out at RT using a rotary evaporator. M-Cyanoborane-2'- Deoxyguanosine 5'-Triphosphate. MCyanoborane-2'-deoxyguanosine (7bdG, 1) was prepared according to the method of Sood et al. (1989, 1992). The method of Ludwig (1981, 1987), as modified by Kovhcs and Otvos (1988), was adapted to the synthesis of M-cyanoborane-2'-deoxyguanosine 5'-triphosphate (5). Proton sponge (214 mg, 1.0 mmol) was added to a solution of 7bdG (153 mg, 0.5 mmol) in trimethyl phosphate (1.25 mL). Phosphoryl chloride (68.2 pL, 0.75 "01) was pipeted into the reaction mixture under vigorous stirring at 0 "C. Stirring was continued with the exclusion of atmospheric
Porter et al. moisture at 0 "C for 2 h. Bis(tri-n-butylammonium) pyrophosphate (0.5 M) in N,N-dimethylformamide (5.0 mL, 2.5 mmol) and tri-n-butylamine (0.5 mL, 2.1 mmol) were added quickly and synchronously to the reaction mixture. The reaction mixture was stirred intensively for 1 min; then aqueous triethylammonium hydrogen carbonate (20 mL, 0.5 M, pH 7.5) was added. Stirring was continued for an additional 1.5 h at RT. The solution was evaporated to dryness. The residue was freed from excess triethylammonium hydrogen carbonate by repeated evaporation with methanol. The residue at evaporation was dissolved in deionized water (5 mL). The solution was applied onto a DEAE-cellulose [HC03-] column (2.1 x 52.0 cm). The column was eluted at 10 "C by using a linear gradient of aqueous triethylammonium hydrogen carbonate, pH 7.5 (2000 mL, 0-0.5 M; speed, 20 mL/fraction/20 min). Appropriate fractions were pooled and evaporated to dryness. Excess triethylammonium hydrogen carbonate was removed from the residue as described above. The evaporated residue was dissolved in deionized water (1 mL). The solution was percolated through a CM-cellulose [Na+] column (1.4 x 12.0 cm). The column was washed with deionized water (speed, 4.0-4.5 mL/lO min). Fractions that showed UV absorbancy were combined and freeze-dried to give 139 mg (40%) of a white solid containing less than 5% contaminants (by thinlayer chromatography on a silica gel chromatoplate in n-PrOWconcentrated N b O W H 2 0 (1 1/7/2), Rf = 0.26, and by 31PNMR): 'H NMR (D20) 6 8.369 (s, lH, H8), 6.6156.145 (unres. lH, Hl'), 4.607-4.600 (unres. lH, H3'), 4.1304.062 (2m, 3H, H4' H5' H5"), 2.608-2.568 and 2.4612.380 (2m, 4H, H2' H2" BHz); 31PNMR (D20) 6 -7.59 (d, JPP= 19.4 Hz, lP, P3), -10.32 (m, Jpp= 19.3 Hz, JPH = 5.5 Hz, lP, Pl), -21.37 (t, J p p = 19.0 Hz, lP, P2); I3C{lH} NMR (D20) 6 155.80 (s, C6), 154.94 (s, C2), 150.72 ( s , C4), 138.75 (s, C8), 110.89 ( s , C5), 86.04 (d, Jcp = 8.6 Hz, C4'), 84.86 ( s , Cl'), 70.42 ( s , C3'), 65.10 (d, Jcp = 5.5 Hz, W ) , 38.63 (s, C2'); MS mlz [ C ~ ~ H I ~ B N ~ O I ~ P ~ N ~ ~ = 635. HPLC. The compound was purified further by HPLC on a Delta-Pak C18 100-8, (3.9 x 300 mm, 15 pm spherical) column (Waters) using a linear gradient of methanol (010% in 10 min) in triethylammonium acetate (0.2 M, pH 7.5) with a flow rate of 4.0 mL/min (retention time, 16.32 min; retention time of 2'-deoxyguanosine 5'-triphosphate, 9.81 min). The HPLC-purified material, after the removal of triethylammonium acetate by lyophilization, was reconverted to the sodium salt as described before. Incorporation of 7bdGTPinto M13mp2. Primer SS20 (5'TATCGGCCTCAGGAAGATCG-3', complementary to positions 6467-6448 of M13mp2; 10 pmol) was 5'-end-labeled with [y3?3]ATP (20 pCi) and polynucleotide kinase (10 units; New England Biolabs) in the manufacturer-supplied buffer (10 pL). The labeled primer was annealed to a singlestranded (+)M13mp2 DNA template (8 pmol) in NT buffer (50 mM Tris-HC1, pH 7.2, 10 mM MgS04, 0.1 mM DTT, and 50 pg/mL BSA; 30 pL) supplemented with dATP, dTTP, dCTP, and either dGTP or 7bdGTP(83 mM each; 7bdGTP €260 = 12.5 x lo3 L/mol.cm) by heating to 95 "C, incubating at 60 "C for 10 min, and then cooling on ice. Sequenase (6.5 units, 0.5 pL) was added, allowed to react for 15 min at room temperature, and stopped by heating at 70 "C for 10 min. The extended primerhemplate duplexes were used directly from the terminated Sequenase reactions.
+ +
+ +
Boronated dGTP: A Good Substrate for DNA Polymerase
Biochemistry, Vol. 34, No. 37, 1995 11965
DMS Cleavage. Extended M13 primerhemplate complexes ( ~ pmol, 1 3.5 pL) were exposed to 20 mM DMS for 10 min on ice. Reactions were stopped by adding DTT to 91 mM. The methylated DNA was cleaved by heating to 90 "C for 8 min and returned to ice. The cleaved DNA was recovered by ethanol precipitation. Restriction Digestion. Extended M 13 primerhemplate complexes (3.5 pL) were digested by HaeZZZ (16 units), PvuZ (10 units), PvuZZ (12 units), or Sau3AZ (4 units) in manufacturer-supplied buffer (20 pL) at 37 "C for 20 min. The DNA was recovered by ethanol precipitation. Electrophoresis. Samples were resuspended in 0.5 x denaturing loading buffer (95% formamide, 0.1% bromophenol blue, 0.1% xylene cyanol, and 20 mM NazEDTA), loaded onto an 8% polyacrylamide/7 M urea gel, and run for 2 h at 75 W in TBE buffer (89 mM Tris-borate and 2 mM Na2EDTA, pH 8.0). The gel was dried under vacuum, and the signal was detected by autoradiography. Quantitation was carried out on a Molecular Dynamics phosphorimager. Polymerase Chain Reaction. Primers used for PCR were SS20 and RP (5'-TCACACAGGAAACAGCTATGAC-3', M13mp2 positions 6200-6221). (+)M13mp2 DNA template (0.5 pmol) was mixed with primers (20 pmol each), dATP, dTTP, dCTP, and either dGTP or 7bdGTP(100 mM each) in 100pL of PCR buffer (50 mM KCl, 1.5 mM MgC12, and 10 mM Tris-HC1, pH 8.3, RT). The reaction mixture was heated to 95 "C for 1 min and returned to ice. Taq polymerase (0.5 pL, 2.5 units) was added, and the PCR was performed in an Ericomp thermal cycler for 25 cycles of 95 "C for 1 min, 56 "C for 1 min, and 72 "C for 1 min. Following PCR amplification, the DNA was ethanol precipitated and then resuspended in 20 p L of 10 mM TrisHC1 (pH 7.6). An aliquot (8 pL) was digested with Sau3AI (4 units) in the manufacturer-supplied buffer (10 pL) for 30 min at 37 "C. Unrestricted (8 pL) and restricted DNA (10 pL taken directly from the restriction digestion) were mixed with 1 p L of glycerol loading buffer (30% glycerol, 0.1% bromophenol blue, and 0.1% xylene cyanol) and separated on a 3% Nu-Sieve (FMC)/l% Sea-Kem (FMC) agarose gel. Kinetic Analysis. The K,,, and V,,, values for the incorporation of normal dGTP and boronated 7bdGTPat 37 "C were determined by a labeled primerhemplate polyacrylamide gel assay (Randall et al., 1987; Boosalis et al., 1987). The system consisted of a radioactively labeled synthetic primer annealed to a synthetic template, which is designed to code for a dG at the first position of primer extension. Primer extension was performed for a range of substrate concentrations. The extension products were separated by denaturing PAGE and quantitated. The initial reaction velocities (vi) could be determined from the integrated band intensities. The intensity of the extended product (ZI)divided by the intensity of the unextended primer (10)plus one-half the intensity of the extended product (0.511) gives a measure of vi (Petruska et al., 1988). Primer (5'-CAGGAACAGCTATGGCCTCA-3'; 30 pmol) was end-labeled with 10 pCi of [Y-~~PIATP, annealed to an equal amount of template (S-GTGTAGCTGAGGCCATAGCTGTTCCTG-3'; 30 pmol), and mixed with 0.109 unit (0.0167 unitheaction) of exonuclease-free Klenow in buffer A (50 mM Tris-HC1, pH 7.6, and 5 mM DTT; 32.5 pL). The primer was extended by mixing 5 p L of the primer/ template duplexes with 5 pL of various concentrations of dGTP or 7bdGTPin buffer B (50 mM Tris-HC1, pH 7.6, 5
mM DTT, and 20 mM MgC12). The reaction was carried out for 1.5 min at 37 "C and stopped by the addition of 10 pL of loading buffer (95% formamide, 20 mM Na2EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanol). The samples were separated on a 16% polyacrylamide/7 M urea sequencing gel, and the intensities of the bands were quantitated. The K,,,and V,, values were calculated from the initial reaction velocities by nonlinear regression analysis. To verify that the initial velocities were obtained at an enzyme-limiting condition, the amount of enzyme was adjusted to produce about 20% extension at the highest substrate concentrations (Goodman et al., 1993). Time Course Reactions. We verified that the initial velocities were obtained during the period of linear accumulation of product by performing a time course experiment for the highest concentrations of substrate. Labeled primerhemplate (30 pmol) and exonuclease-free Klenow (0.109 unit) in buffer A (32.5 pL) were mixed with dGTP (0.2 pM) or 7bdGTP(0.1 pM) in buffer B (32.5 p L ) at 37 "C. Aliquots were withdrawn at 30-s intervals and mixed with loading buffer. Analysis showed that the product accumulated linearly from 30 s to 3 min (1-2 = 0.994 for dGTP; 9 = 0.996 for 7bdGTP).
RESULTS Once pure N"-cyanoborane-2'-deoxyguanosine 5'-triphosphate (5, 7bdGTP)was prepared, two questions had to be answered to determine whether the 7bdGTPwas a substrate for DNA polymerase. (1) Could a primer be extended in the presence of 7bdGTP? (2) Could the 7bdG moiety be incorporated stably into the newly synthesized duplex? To answer the first question, an oligonucleotide primer was extended with DNA polymerase in the presence of dATP, dCTP, dTTP, and either dGTP or 7bdGTP. Results, described here, show that the primer could be extended by Sequenase using 7bdGTPfor a substrate. To answer the second question, the presence of the 7bdGresidue in the newly synthesized duplex was verified by a methylation protection experiment and several restriction enzyme protection experiments. When 7bdGTPwas used for a substrate, results show that (a) the DNA product was protected from cleavage by treatment with dimethyl sulfate (DMS) and heat, and (b) the dG-containing recognition sites in the product were protected from cleavage by the restriction enzymes HaeIII, PvuI, PvuII, and Sau3AI. Incorporation of 7bdGTP by Sequenase. The labeled primer, SS20, was annealed to the (+) strand of M13mp2 and extended by Sequenase in the presence of dATP, dCTP, dTTP, and either dGTP (lanes labeled G) or 7bdGTP(lanes labeled b). Figure 1 shows that the SS20 primer was extended in the presence of dGTP (lane 1) as well as 7bdGTP (lane 2). The dGTP products extended well into the upper ranges of the gel (500 to several thousand base pairs; lane 1). Likewise, the 7bdGTP (lane 2) was incorporated into duplexes extending to hundreds of base pairs, indicating multiple incorporations and extensions of the boronated deoxynucleoside triphosphate. However, the pattern of extension products shown in lane 2 differed somewhat from the pattem produced by normal dGTP primer extension, in that there were pronounced dark bands at positions that required three or more consecutive 7bdGTPincorporations. For example, there are three intense groups of bands at position 6356 (GGG), at position 6343 (GGG), and especially
11966 Biochemistry, Vol. 34, No. 37, I995 Cont'l heat
DMS
Porter et al. incorporated a normal dGTP that could be present at a low concentration due to contamination in the starting material or deboronation of 7bdGTP. To confirm that the 7bdGTPhad been incorporated stably into the extension products, we performed methylation protection and restriction enzyme protection assays, which are described in the following sections.
Haelll PvulI Pvul Sau3Al
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n n n n
G b G b G b 1 2 3 4 5 6
G b G b G b G b 7 8 9 10 11 12 1314
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6300 6340 6355
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